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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

Fragment encompassing aa 37–191 of HCV core is responsible for the activation of ER oxidoreductin 1α (Ero1α) which contributes to the production of hydrogen peroxide. (A,B) Induction of the expression of Ero1α assessed by RT-qPCR (A) and Western-blotting (B) of Huh7 cells 40 h post transfection with plasmids expressing HCV core; (C,D) Silencing of Ero1α expression in naïve and HCV core-expressing Huh7 cells. Respective cells were transfected with anti-Ero1α siRNA or mock siRNA as a negative control; expression of Ero1α was analyzed by RT-qPCR (C), and for HCV core expressing cells also by Western-blotting (D), values were normalized to the expression of β-actin; (E–G) Silencing of Ero1α in HCV core expressing Huh7 cells using specific siRNA (siEro1a) inhibits the production of hydrogen peroxide; no effect is observed after treatment of cells with mock siRNA (siMock). To assess this, Huh7 cells were transfected with the respective siRNA and a mixture of plasmids expressing core(1–191) and HyPer-cyto sensor; mean fold increase in HyPer-cyto fluorescence (E) and the % of fluorescent cells (F) was quantified by flow cytometry. Alternatively, Huh7 cells were transfected with the plasmid expressing core(1–191) and a mixture of respective siRNA; the induction of ROS was assessed by the DCFH assay (G). Fluorescence was expressed as a fold-increase compared to the fluorescence measured in mock-treated Huh7 cells transfected with the empty vector pVax1. For experimental details, see Materials and Methods. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
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viruses-07-02745-f005: Fragment encompassing aa 37–191 of HCV core is responsible for the activation of ER oxidoreductin 1α (Ero1α) which contributes to the production of hydrogen peroxide. (A,B) Induction of the expression of Ero1α assessed by RT-qPCR (A) and Western-blotting (B) of Huh7 cells 40 h post transfection with plasmids expressing HCV core; (C,D) Silencing of Ero1α expression in naïve and HCV core-expressing Huh7 cells. Respective cells were transfected with anti-Ero1α siRNA or mock siRNA as a negative control; expression of Ero1α was analyzed by RT-qPCR (C), and for HCV core expressing cells also by Western-blotting (D), values were normalized to the expression of β-actin; (E–G) Silencing of Ero1α in HCV core expressing Huh7 cells using specific siRNA (siEro1a) inhibits the production of hydrogen peroxide; no effect is observed after treatment of cells with mock siRNA (siMock). To assess this, Huh7 cells were transfected with the respective siRNA and a mixture of plasmids expressing core(1–191) and HyPer-cyto sensor; mean fold increase in HyPer-cyto fluorescence (E) and the % of fluorescent cells (F) was quantified by flow cytometry. Alternatively, Huh7 cells were transfected with the plasmid expressing core(1–191) and a mixture of respective siRNA; the induction of ROS was assessed by the DCFH assay (G). Fluorescence was expressed as a fold-increase compared to the fluorescence measured in mock-treated Huh7 cells transfected with the empty vector pVax1. For experimental details, see Materials and Methods. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.

Mentions: In an independent set of experiments, we tested whether the N-terminally deleted core could also induce other ROS types, namely hydrogen peroxide. We have demonstrated that the major source of H2O2 production in cells expressing HCV core is located outside mitochondria (Figure 2). One of the key sources of hydrogen peroxide outside mitochondria is the ER oxidoreductin 1α (Ero1α) [19]. RT-qPCR and Western blot analysis demonstrated that both the full-length and the N-terminally deleted core variants induce the expression of Ero1α (Figure 5A,B). To estimate the impact of Ero1α on the production of ROS, we silenced its expression with siRNA. The level of Ero1α in the “naïve” Huh7 cells transfected with Ero1α-specific siRNA decreased 30 times, and in the HCV core expressing Huh7 cells, 12 times, compared to the respective cells treated with mock siRNA (Figure 5С). Suppression of the expression of Ero1α coincided with a significant reduction of the production of H2O2, as was registered in the transfection of Ero1α-silenced core-expressing Huh7 cells with a plasmid encoding HyPer-Cyto (Figure 5E). It is noteworthy that treatment with Ero1α-specific siRNA affected only the levels of fluorescence (Figure 5E), not the number of fluorescent cells (Figure 5F). The latter indicated that Ero1α silencing had a specific effect on the ROS-producing/fluorescent cell population. A similar result was obtained in the DCFH assay (Figure 5G). Overall, this demonstrated that the N-terminally deleted core protein can trigger both the production of superoxide anion mediated by CYP2E1 and the production of H2O2 mediated by Ero1α.


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)

Fragment encompassing aa 37–191 of HCV core is responsible for the activation of ER oxidoreductin 1α (Ero1α) which contributes to the production of hydrogen peroxide. (A,B) Induction of the expression of Ero1α assessed by RT-qPCR (A) and Western-blotting (B) of Huh7 cells 40 h post transfection with plasmids expressing HCV core; (C,D) Silencing of Ero1α expression in naïve and HCV core-expressing Huh7 cells. Respective cells were transfected with anti-Ero1α siRNA or mock siRNA as a negative control; expression of Ero1α was analyzed by RT-qPCR (C), and for HCV core expressing cells also by Western-blotting (D), values were normalized to the expression of β-actin; (E–G) Silencing of Ero1α in HCV core expressing Huh7 cells using specific siRNA (siEro1a) inhibits the production of hydrogen peroxide; no effect is observed after treatment of cells with mock siRNA (siMock). To assess this, Huh7 cells were transfected with the respective siRNA and a mixture of plasmids expressing core(1–191) and HyPer-cyto sensor; mean fold increase in HyPer-cyto fluorescence (E) and the % of fluorescent cells (F) was quantified by flow cytometry. Alternatively, Huh7 cells were transfected with the plasmid expressing core(1–191) and a mixture of respective siRNA; the induction of ROS was assessed by the DCFH assay (G). Fluorescence was expressed as a fold-increase compared to the fluorescence measured in mock-treated Huh7 cells transfected with the empty vector pVax1. For experimental details, see Materials and Methods. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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viruses-07-02745-f005: Fragment encompassing aa 37–191 of HCV core is responsible for the activation of ER oxidoreductin 1α (Ero1α) which contributes to the production of hydrogen peroxide. (A,B) Induction of the expression of Ero1α assessed by RT-qPCR (A) and Western-blotting (B) of Huh7 cells 40 h post transfection with plasmids expressing HCV core; (C,D) Silencing of Ero1α expression in naïve and HCV core-expressing Huh7 cells. Respective cells were transfected with anti-Ero1α siRNA or mock siRNA as a negative control; expression of Ero1α was analyzed by RT-qPCR (C), and for HCV core expressing cells also by Western-blotting (D), values were normalized to the expression of β-actin; (E–G) Silencing of Ero1α in HCV core expressing Huh7 cells using specific siRNA (siEro1a) inhibits the production of hydrogen peroxide; no effect is observed after treatment of cells with mock siRNA (siMock). To assess this, Huh7 cells were transfected with the respective siRNA and a mixture of plasmids expressing core(1–191) and HyPer-cyto sensor; mean fold increase in HyPer-cyto fluorescence (E) and the % of fluorescent cells (F) was quantified by flow cytometry. Alternatively, Huh7 cells were transfected with the plasmid expressing core(1–191) and a mixture of respective siRNA; the induction of ROS was assessed by the DCFH assay (G). Fluorescence was expressed as a fold-increase compared to the fluorescence measured in mock-treated Huh7 cells transfected with the empty vector pVax1. For experimental details, see Materials and Methods. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
Mentions: In an independent set of experiments, we tested whether the N-terminally deleted core could also induce other ROS types, namely hydrogen peroxide. We have demonstrated that the major source of H2O2 production in cells expressing HCV core is located outside mitochondria (Figure 2). One of the key sources of hydrogen peroxide outside mitochondria is the ER oxidoreductin 1α (Ero1α) [19]. RT-qPCR and Western blot analysis demonstrated that both the full-length and the N-terminally deleted core variants induce the expression of Ero1α (Figure 5A,B). To estimate the impact of Ero1α on the production of ROS, we silenced its expression with siRNA. The level of Ero1α in the “naïve” Huh7 cells transfected with Ero1α-specific siRNA decreased 30 times, and in the HCV core expressing Huh7 cells, 12 times, compared to the respective cells treated with mock siRNA (Figure 5С). Suppression of the expression of Ero1α coincided with a significant reduction of the production of H2O2, as was registered in the transfection of Ero1α-silenced core-expressing Huh7 cells with a plasmid encoding HyPer-Cyto (Figure 5E). It is noteworthy that treatment with Ero1α-specific siRNA affected only the levels of fluorescence (Figure 5E), not the number of fluorescent cells (Figure 5F). The latter indicated that Ero1α silencing had a specific effect on the ROS-producing/fluorescent cell population. A similar result was obtained in the DCFH assay (Figure 5G). Overall, this demonstrated that the N-terminally deleted core protein can trigger both the production of superoxide anion mediated by CYP2E1 and the production of H2O2 mediated by Ero1α.

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