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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 residues 37–191 of HCV core contributes to ROS production by activating the expression of cytochrome P450 2E1 (CYP2E1). (A,B) Up-regulation of CYP2E1 expression in Huh7 cells transfected with plasmids expressing core(1–191) and core(37–191) but not core(1–36) or NS5B protein control assessed by the real-time-qPCR (A) and Western blotting (B); The levels of CYP2E1 mRNA assessed by RT-qPCR (see Materials and Methods) are represented as relative to the expression of β-actin; the resultant values are further normalized to the relative expression of CYP2E1 in Huh7 cells transfected with the empty vector pVax1; (C,D) CYP2E1 inhibitor 4-methylpyrazole (4-MP) suppressed the production of superoxide anion (C) and ROS (D) in Huh7 cells expressing the full-length core(1–191) and the N-terminally truncated variant core(37–191). Fluorescence levels are expressed as a fold-increase compared to the mock-treated Huh7 cells transfected with the empty vector pVax1. All data represent the means ± S.D. from the triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
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viruses-07-02745-f004: Fragment encompassing aa residues 37–191 of HCV core contributes to ROS production by activating the expression of cytochrome P450 2E1 (CYP2E1). (A,B) Up-regulation of CYP2E1 expression in Huh7 cells transfected with plasmids expressing core(1–191) and core(37–191) but not core(1–36) or NS5B protein control assessed by the real-time-qPCR (A) and Western blotting (B); The levels of CYP2E1 mRNA assessed by RT-qPCR (see Materials and Methods) are represented as relative to the expression of β-actin; the resultant values are further normalized to the relative expression of CYP2E1 in Huh7 cells transfected with the empty vector pVax1; (C,D) CYP2E1 inhibitor 4-methylpyrazole (4-MP) suppressed the production of superoxide anion (C) and ROS (D) in Huh7 cells expressing the full-length core(1–191) and the N-terminally truncated variant core(37–191). Fluorescence levels are expressed as a fold-increase compared to the mock-treated Huh7 cells transfected with the empty vector pVax1. All data represent the means ± S.D. from the triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.

Mentions: We have next examined whether the induction of oxidative stress by HCV core protein could be mediated by ROS-generating enzymes other than NADPH oxidases. One of the candidate inducers of ROS is a cytochrome P450 isoform 2E1 (CYP2E1) [40,41]. Indeed, we found that expression of the full-length core and core(37–191) resulted in a substantial increase in the mRNA and protein levels of CYP2E1 (Figure 4A,B). No significant up-regulation was observed upon the expression of core(1–36) or NS5B control. A two-fold reduction of ROS and superoxide anion levels in core(1–191)- and core(37–191)-expressing cells was observed upon treatment of expressing cells with a specific inhibitor of CYP2E1 activity 4-methylpyrazole (4-MP) [42] (Figure 4C,D). As in the case of NOX1 and NOX4, siRNA silencing of CYP2E1 in HCV core-expressing Huh7 cells also resulted in a decrease in the levels of ROS in the DCFH-, and of the superoxide anion in the DHE-based assay (data not shown). Thus, cytochrome P450 isoform 2E1 triggers the production in Huh7 cells of superoxide anion (and possibly of the other types of ROS).


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 residues 37–191 of HCV core contributes to ROS production by activating the expression of cytochrome P450 2E1 (CYP2E1). (A,B) Up-regulation of CYP2E1 expression in Huh7 cells transfected with plasmids expressing core(1–191) and core(37–191) but not core(1–36) or NS5B protein control assessed by the real-time-qPCR (A) and Western blotting (B); The levels of CYP2E1 mRNA assessed by RT-qPCR (see Materials and Methods) are represented as relative to the expression of β-actin; the resultant values are further normalized to the relative expression of CYP2E1 in Huh7 cells transfected with the empty vector pVax1; (C,D) CYP2E1 inhibitor 4-methylpyrazole (4-MP) suppressed the production of superoxide anion (C) and ROS (D) in Huh7 cells expressing the full-length core(1–191) and the N-terminally truncated variant core(37–191). Fluorescence levels are expressed as a fold-increase compared to the mock-treated Huh7 cells transfected with the empty vector pVax1. All data represent the means ± S.D. from the triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488712&req=5

viruses-07-02745-f004: Fragment encompassing aa residues 37–191 of HCV core contributes to ROS production by activating the expression of cytochrome P450 2E1 (CYP2E1). (A,B) Up-regulation of CYP2E1 expression in Huh7 cells transfected with plasmids expressing core(1–191) and core(37–191) but not core(1–36) or NS5B protein control assessed by the real-time-qPCR (A) and Western blotting (B); The levels of CYP2E1 mRNA assessed by RT-qPCR (see Materials and Methods) are represented as relative to the expression of β-actin; the resultant values are further normalized to the relative expression of CYP2E1 in Huh7 cells transfected with the empty vector pVax1; (C,D) CYP2E1 inhibitor 4-methylpyrazole (4-MP) suppressed the production of superoxide anion (C) and ROS (D) in Huh7 cells expressing the full-length core(1–191) and the N-terminally truncated variant core(37–191). Fluorescence levels are expressed as a fold-increase compared to the mock-treated Huh7 cells transfected with the empty vector pVax1. All data represent the means ± S.D. from the triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
Mentions: We have next examined whether the induction of oxidative stress by HCV core protein could be mediated by ROS-generating enzymes other than NADPH oxidases. One of the candidate inducers of ROS is a cytochrome P450 isoform 2E1 (CYP2E1) [40,41]. Indeed, we found that expression of the full-length core and core(37–191) resulted in a substantial increase in the mRNA and protein levels of CYP2E1 (Figure 4A,B). No significant up-regulation was observed upon the expression of core(1–36) or NS5B control. A two-fold reduction of ROS and superoxide anion levels in core(1–191)- and core(37–191)-expressing cells was observed upon treatment of expressing cells with a specific inhibitor of CYP2E1 activity 4-methylpyrazole (4-MP) [42] (Figure 4C,D). As in the case of NOX1 and NOX4, siRNA silencing of CYP2E1 in HCV core-expressing Huh7 cells also resulted in a decrease in the levels of ROS in the DCFH-, and of the superoxide anion in the DHE-based assay (data not shown). Thus, cytochrome P450 isoform 2E1 triggers the production in Huh7 cells of superoxide anion (and possibly of the other types of ROS).

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