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

Alterations in the architecture of HeLa cells following expression of HCV proteins from VT7-HCV7.9 seen by electron microscopy. HeLa 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 as described under Materials and Methods.A: Cellular architecture of uninfected HeLa 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 and D: A general view of cells infected with VT7-HCV7.9 in the presence of IPTG, showing few IVs, large EDS, swollen mitochondria and vacuoles. E: High magnification of infected VT7-HCV7.9 cells in the presence of IPTG showing EDS with membranes, TS and swollen mitochondria with a protruding membrane. Note: Nucleus (N), mitochondria (m), Golgi apparatus (G), immature virus (IV), intracellular mature virus (IMV), tubular structures (TS), electron dense structures in membranous webs (EDS). Bar: 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Alterations in the architecture of HeLa cells following expression of HCV proteins from VT7-HCV7.9 seen by electron microscopy. HeLa 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 as described under Materials and Methods.A: Cellular architecture of uninfected HeLa 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 and D: A general view of cells infected with VT7-HCV7.9 in the presence of IPTG, showing few IVs, large EDS, swollen mitochondria and vacuoles. E: High magnification of infected VT7-HCV7.9 cells in the presence of IPTG showing EDS with membranes, TS and swollen mitochondria with a protruding membrane. Note: Nucleus (N), mitochondria (m), Golgi apparatus (G), immature virus (IV), intracellular mature virus (IMV), tubular structures (TS), electron dense structures in membranous webs (EDS). Bar: 500 nm.

Mentions: To gain more detail information on the subcellular changes induced by HCV polyprotein expression, we performed transmission electron microscopy (EM) analysis. 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 collected and ultrathin sections visualized by EM at low and high magnification. While in cells infected with VT7-HCV7.9, in the absence of IPTG there are high number of immature (IVs) and intracellular mature (IMVs) forms of VACV virus, in cells infected with VT7-HCV7.9 in the presence of IPTG fewer IVs and IMVs were observed, corroborating our previous finding that the expression of HCV proteins blocked VACV morphogenesis [12]. In addition, several morphological alterations were detected in cells expressing the HCV polyprotein when compared with uninfected cells (Fig. 2A) or with cells only expressing VACV proteins (Fig. 2B). The first alteration seen was the loss of spatial organization of the ER, with vesicles embedded in a membrane matrix of circular or tightly undulating membranes, forming electron dense structures indicated as EDS (Fig. 2C and 2D). These cytoplasmic structures resemble those structures called "membraneous webs" that have been visualized in human hepatoma Huh7 cells expressing a subgenomic HCV replicon [5,14,15]. Other alterations observed were the presence of vacuoles (indicated as V) often surrounding the compact structures, as well as the presence of swollen mitochondria (indicated as m) (Fig. 2D and 2E). Higher magnification of the electron dense structures in cells expressing the HCV polyprotein revealed membranes and tubular structures (indicated as TS) as part of the EDS (Fig. 2E).


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)

Alterations in the architecture of HeLa cells following expression of HCV proteins from VT7-HCV7.9 seen by electron microscopy. HeLa 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 as described under Materials and Methods.A: Cellular architecture of uninfected HeLa 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 and D: A general view of cells infected with VT7-HCV7.9 in the presence of IPTG, showing few IVs, large EDS, swollen mitochondria and vacuoles. E: High magnification of infected VT7-HCV7.9 cells in the presence of IPTG showing EDS with membranes, TS and swollen mitochondria with a protruding membrane. Note: Nucleus (N), mitochondria (m), Golgi apparatus (G), immature virus (IV), intracellular mature virus (IMV), tubular structures (TS), electron dense structures in membranous webs (EDS). Bar: 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Alterations in the architecture of HeLa cells following expression of HCV proteins from VT7-HCV7.9 seen by electron microscopy. HeLa 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 as described under Materials and Methods.A: Cellular architecture of uninfected HeLa 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 and D: A general view of cells infected with VT7-HCV7.9 in the presence of IPTG, showing few IVs, large EDS, swollen mitochondria and vacuoles. E: High magnification of infected VT7-HCV7.9 cells in the presence of IPTG showing EDS with membranes, TS and swollen mitochondria with a protruding membrane. Note: Nucleus (N), mitochondria (m), Golgi apparatus (G), immature virus (IV), intracellular mature virus (IMV), tubular structures (TS), electron dense structures in membranous webs (EDS). Bar: 500 nm.
Mentions: To gain more detail information on the subcellular changes induced by HCV polyprotein expression, we performed transmission electron microscopy (EM) analysis. 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 collected and ultrathin sections visualized by EM at low and high magnification. While in cells infected with VT7-HCV7.9, in the absence of IPTG there are high number of immature (IVs) and intracellular mature (IMVs) forms of VACV virus, in cells infected with VT7-HCV7.9 in the presence of IPTG fewer IVs and IMVs were observed, corroborating our previous finding that the expression of HCV proteins blocked VACV morphogenesis [12]. In addition, several morphological alterations were detected in cells expressing the HCV polyprotein when compared with uninfected cells (Fig. 2A) or with cells only expressing VACV proteins (Fig. 2B). The first alteration seen was the loss of spatial organization of the ER, with vesicles embedded in a membrane matrix of circular or tightly undulating membranes, forming electron dense structures indicated as EDS (Fig. 2C and 2D). These cytoplasmic structures resemble those structures called "membraneous webs" that have been visualized in human hepatoma Huh7 cells expressing a subgenomic HCV replicon [5,14,15]. Other alterations observed were the presence of vacuoles (indicated as V) often surrounding the compact structures, as well as the presence of swollen mitochondria (indicated as m) (Fig. 2D and 2E). Higher magnification of the electron dense structures in cells expressing the HCV polyprotein revealed membranes and tubular structures (indicated as TS) as part of the EDS (Fig. 2E).

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