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HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma Huh7 cells.

Ivanov AV, Smirnova OA, Petrushanko IY, Ivanova ON, Karpenko IL, Alekseeva E, Sominskaya I, Makarov AA, Bartosch B, Kochetkov SN, Isaguliants MG - Viruses (2015)

Bottom Line: Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\).Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production.Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. aivanov@yandex.ru.

ABSTRACT
Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37-191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

No MeSH data available.


Related in: MedlinePlus

Expression of fragments encompassing HCV core amino acids 1–36 and 37–191 leads to the enhanced production of superoxide anion and hydrogen peroxide. (A–C) Huh7 cells were seeded on 24-well plates, transfected with plasmids encoding the full-length or truncated forms of HCV core, and 28 h posttransfection stained with one of the dyes: ROS-unspecific 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA) (A), superoxide anion-specific dihydroethidium (DHE) (B), or a mitochondrially-targeted superoxide-specific MitoSox Red (C), Fluorescence intensity was measured using the Chameleon V microplate reader. Signal was normalized to the levels of fluorescence in Huh7 cells transfected with the empty vector pVax1; (D–I) Huh7 cells were seeded on 6-well (D,E,G,H) or 24-well (F,I) plates, co-transfected with the plasmid expressing a variant of HCV core and the reporter plasmid expressing a cytoplasmic hydrogen peroxide sensor HyPer-cyto (D–F) or a mitochondrial hydrogen peroxide sensor HyPer-dMito (G–I). Percent cells with the elevated level of fluorescence (D,G), and the relative increase in total fluorescence (E,H) as compared to untransfected Huh7 cells, were evaluated by flow cytometry. HyPer fluorescence was also measured on a microplate reader at 535 nm after excitation independently at 485 and 416 nm (F,I). Control samples were treated with H2O2 or N-acetylcysteine (NAC). Values on each diagram are means ± S.D. of the triplicate measurements done in three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001.
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viruses-07-02745-f002: Expression of fragments encompassing HCV core amino acids 1–36 and 37–191 leads to the enhanced production of superoxide anion and hydrogen peroxide. (A–C) Huh7 cells were seeded on 24-well plates, transfected with plasmids encoding the full-length or truncated forms of HCV core, and 28 h posttransfection stained with one of the dyes: ROS-unspecific 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA) (A), superoxide anion-specific dihydroethidium (DHE) (B), or a mitochondrially-targeted superoxide-specific MitoSox Red (C), Fluorescence intensity was measured using the Chameleon V microplate reader. Signal was normalized to the levels of fluorescence in Huh7 cells transfected with the empty vector pVax1; (D–I) Huh7 cells were seeded on 6-well (D,E,G,H) or 24-well (F,I) plates, co-transfected with the plasmid expressing a variant of HCV core and the reporter plasmid expressing a cytoplasmic hydrogen peroxide sensor HyPer-cyto (D–F) or a mitochondrial hydrogen peroxide sensor HyPer-dMito (G–I). Percent cells with the elevated level of fluorescence (D,G), and the relative increase in total fluorescence (E,H) as compared to untransfected Huh7 cells, were evaluated by flow cytometry. HyPer fluorescence was also measured on a microplate reader at 535 nm after excitation independently at 485 and 416 nm (F,I). Control samples were treated with H2O2 or N-acetylcysteine (NAC). Values on each diagram are means ± S.D. of the triplicate measurements done in three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: ROS levels in cells expressing core fragments were quantified using 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA). DCFHDA penetrates the cells, where it is de-esterified into DCFH; the latter is oxidized by different types of ROS into specific fluorescent products [38]. Both core(1–36) and core(37–191) variants induced a significant elevation of ROS levels (2.6- and 3.6-fold, respectively; Figure 2A), and together added up to the effect of the full-length protein.


HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma Huh7 cells.

Ivanov AV, Smirnova OA, Petrushanko IY, Ivanova ON, Karpenko IL, Alekseeva E, Sominskaya I, Makarov AA, Bartosch B, Kochetkov SN, Isaguliants MG - Viruses (2015)

Expression of fragments encompassing HCV core amino acids 1–36 and 37–191 leads to the enhanced production of superoxide anion and hydrogen peroxide. (A–C) Huh7 cells were seeded on 24-well plates, transfected with plasmids encoding the full-length or truncated forms of HCV core, and 28 h posttransfection stained with one of the dyes: ROS-unspecific 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA) (A), superoxide anion-specific dihydroethidium (DHE) (B), or a mitochondrially-targeted superoxide-specific MitoSox Red (C), Fluorescence intensity was measured using the Chameleon V microplate reader. Signal was normalized to the levels of fluorescence in Huh7 cells transfected with the empty vector pVax1; (D–I) Huh7 cells were seeded on 6-well (D,E,G,H) or 24-well (F,I) plates, co-transfected with the plasmid expressing a variant of HCV core and the reporter plasmid expressing a cytoplasmic hydrogen peroxide sensor HyPer-cyto (D–F) or a mitochondrial hydrogen peroxide sensor HyPer-dMito (G–I). Percent cells with the elevated level of fluorescence (D,G), and the relative increase in total fluorescence (E,H) as compared to untransfected Huh7 cells, were evaluated by flow cytometry. HyPer fluorescence was also measured on a microplate reader at 535 nm after excitation independently at 485 and 416 nm (F,I). Control samples were treated with H2O2 or N-acetylcysteine (NAC). Values on each diagram are means ± S.D. of the triplicate measurements done in three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488712&req=5

viruses-07-02745-f002: Expression of fragments encompassing HCV core amino acids 1–36 and 37–191 leads to the enhanced production of superoxide anion and hydrogen peroxide. (A–C) Huh7 cells were seeded on 24-well plates, transfected with plasmids encoding the full-length or truncated forms of HCV core, and 28 h posttransfection stained with one of the dyes: ROS-unspecific 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA) (A), superoxide anion-specific dihydroethidium (DHE) (B), or a mitochondrially-targeted superoxide-specific MitoSox Red (C), Fluorescence intensity was measured using the Chameleon V microplate reader. Signal was normalized to the levels of fluorescence in Huh7 cells transfected with the empty vector pVax1; (D–I) Huh7 cells were seeded on 6-well (D,E,G,H) or 24-well (F,I) plates, co-transfected with the plasmid expressing a variant of HCV core and the reporter plasmid expressing a cytoplasmic hydrogen peroxide sensor HyPer-cyto (D–F) or a mitochondrial hydrogen peroxide sensor HyPer-dMito (G–I). Percent cells with the elevated level of fluorescence (D,G), and the relative increase in total fluorescence (E,H) as compared to untransfected Huh7 cells, were evaluated by flow cytometry. HyPer fluorescence was also measured on a microplate reader at 535 nm after excitation independently at 485 and 416 nm (F,I). Control samples were treated with H2O2 or N-acetylcysteine (NAC). Values on each diagram are means ± S.D. of the triplicate measurements done in three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: ROS levels in cells expressing core fragments were quantified using 2',7'-dichlorodihydrofluoresceine diacetate (DCFHDA). DCFHDA penetrates the cells, where it is de-esterified into DCFH; the latter is oxidized by different types of ROS into specific fluorescent products [38]. Both core(1–36) and core(37–191) variants induced a significant elevation of ROS levels (2.6- and 3.6-fold, respectively; Figure 2A), and together added up to the effect of the full-length protein.

Bottom Line: Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\).Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production.Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. aivanov@yandex.ru.

ABSTRACT
Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\(\upbeta\)1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37-191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\(\upalpha\). The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

No MeSH data available.


Related in: MedlinePlus