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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

The N-terminus of HCV core protein triggers oxidative stress by independently inducing the expression of NOX1 and NOX4. (A–E) Quantification in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) of the mRNA levels of NOX1 (A), NOX4 (B), TGFβ1 (D), and COX-2 (E); (C,F) Up-regulation of the translation of NOX1, NOX4 (C), and COX-2 (F) in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) revealed by SDS-PAGE of the respective cell lysates with subsequent Western blotting using antibodies specific to NOX1, NOX4, or COX-2 (as depicted to the right); (G) Absence of co-regulation of expression of NOX1, NOX4, and COX-2 demonstrated by a selective inhibition of their expression in Huh7 cells expressing core(1–191) by the specific siRNA (given at the top); (H–J) siRNAs ensured an effective suppression of expression of the respective protein as revealed by Western blot analysis; (K,L) DCFH (K) and DHE (L) staining reveals a partial inhibition of ROS production induced by core(1–191) and core(1–36) in Huh7 cells after silencing of expression of NOX1, or NOX4, or COX-2 with specific siRNAs; (M,N) Suppression of the expression of NOX1 and NOX4 (M) and of superoxide anion production (N) in Huh7 cells expressing core(1–191) by antibodies specific to TGFβ1 (see Materials and Methods section for experimental details). Huh7 cells transfected with the empty pVax1 vector or pVax1 expressing NS5B served as negative controls. Levels of NOX1, NOX4, TGFβ1, or COX-2 mRNA were first expressed as relative to the content in the sample of mRNA of β-actin, and then normalized to the relative content of the respective mRNA in Huh7 cells transfected with the empty vector pVax1. Levels of superoxde anion were measured using DHE. 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-f003: The N-terminus of HCV core protein triggers oxidative stress by independently inducing the expression of NOX1 and NOX4. (A–E) Quantification in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) of the mRNA levels of NOX1 (A), NOX4 (B), TGFβ1 (D), and COX-2 (E); (C,F) Up-regulation of the translation of NOX1, NOX4 (C), and COX-2 (F) in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) revealed by SDS-PAGE of the respective cell lysates with subsequent Western blotting using antibodies specific to NOX1, NOX4, or COX-2 (as depicted to the right); (G) Absence of co-regulation of expression of NOX1, NOX4, and COX-2 demonstrated by a selective inhibition of their expression in Huh7 cells expressing core(1–191) by the specific siRNA (given at the top); (H–J) siRNAs ensured an effective suppression of expression of the respective protein as revealed by Western blot analysis; (K,L) DCFH (K) and DHE (L) staining reveals a partial inhibition of ROS production induced by core(1–191) and core(1–36) in Huh7 cells after silencing of expression of NOX1, or NOX4, or COX-2 with specific siRNAs; (M,N) Suppression of the expression of NOX1 and NOX4 (M) and of superoxide anion production (N) in Huh7 cells expressing core(1–191) by antibodies specific to TGFβ1 (see Materials and Methods section for experimental details). Huh7 cells transfected with the empty pVax1 vector or pVax1 expressing NS5B served as negative controls. Levels of NOX1, NOX4, TGFβ1, or COX-2 mRNA were first expressed as relative to the content in the sample of mRNA of β-actin, and then normalized to the relative content of the respective mRNA in Huh7 cells transfected with the empty vector pVax1. Levels of superoxde anion were measured using DHE. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.

Mentions: Our next step was to identify the molecular mechanisms by which HCV core induces the production of superoxide anion. In the cytoplasm, superoxide anion is generated by various enzymes, including the NOX family of NADPH oxidases. Crucial sources of superoxide anion in HCV-infected hepatocytes are NADPH oxidases 1 and 4 [21,22]. With this in mind, we assessed the effects of HCV core variants on the level of expression of NOX1 and NOX4 using RT-qPCR and Western blot analysis. A significant increase in both NOX1 and NOX4 mRNA and protein levels was observed in Huh7 cells expressing the full-length core(1–191) protein as well as core variants core(1–151) and core(1–36) (Figure 3, panels A–C). No up-regulation was observed in cells expressing core(37–191) or NS5B control (Figure 3A–C).


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)

The N-terminus of HCV core protein triggers oxidative stress by independently inducing the expression of NOX1 and NOX4. (A–E) Quantification in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) of the mRNA levels of NOX1 (A), NOX4 (B), TGFβ1 (D), and COX-2 (E); (C,F) Up-regulation of the translation of NOX1, NOX4 (C), and COX-2 (F) in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) revealed by SDS-PAGE of the respective cell lysates with subsequent Western blotting using antibodies specific to NOX1, NOX4, or COX-2 (as depicted to the right); (G) Absence of co-regulation of expression of NOX1, NOX4, and COX-2 demonstrated by a selective inhibition of their expression in Huh7 cells expressing core(1–191) by the specific siRNA (given at the top); (H–J) siRNAs ensured an effective suppression of expression of the respective protein as revealed by Western blot analysis; (K,L) DCFH (K) and DHE (L) staining reveals a partial inhibition of ROS production induced by core(1–191) and core(1–36) in Huh7 cells after silencing of expression of NOX1, or NOX4, or COX-2 with specific siRNAs; (M,N) Suppression of the expression of NOX1 and NOX4 (M) and of superoxide anion production (N) in Huh7 cells expressing core(1–191) by antibodies specific to TGFβ1 (see Materials and Methods section for experimental details). Huh7 cells transfected with the empty pVax1 vector or pVax1 expressing NS5B served as negative controls. Levels of NOX1, NOX4, TGFβ1, or COX-2 mRNA were first expressed as relative to the content in the sample of mRNA of β-actin, and then normalized to the relative content of the respective mRNA in Huh7 cells transfected with the empty vector pVax1. Levels of superoxde anion were measured using DHE. 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-f003: The N-terminus of HCV core protein triggers oxidative stress by independently inducing the expression of NOX1 and NOX4. (A–E) Quantification in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) of the mRNA levels of NOX1 (A), NOX4 (B), TGFβ1 (D), and COX-2 (E); (C,F) Up-regulation of the translation of NOX1, NOX4 (C), and COX-2 (F) in Huh7 cells expressing the full length core(1–191) or truncated forms of HCV core protein core(1–36), core(37–191) or core(1–151) revealed by SDS-PAGE of the respective cell lysates with subsequent Western blotting using antibodies specific to NOX1, NOX4, or COX-2 (as depicted to the right); (G) Absence of co-regulation of expression of NOX1, NOX4, and COX-2 demonstrated by a selective inhibition of their expression in Huh7 cells expressing core(1–191) by the specific siRNA (given at the top); (H–J) siRNAs ensured an effective suppression of expression of the respective protein as revealed by Western blot analysis; (K,L) DCFH (K) and DHE (L) staining reveals a partial inhibition of ROS production induced by core(1–191) and core(1–36) in Huh7 cells after silencing of expression of NOX1, or NOX4, or COX-2 with specific siRNAs; (M,N) Suppression of the expression of NOX1 and NOX4 (M) and of superoxide anion production (N) in Huh7 cells expressing core(1–191) by antibodies specific to TGFβ1 (see Materials and Methods section for experimental details). Huh7 cells transfected with the empty pVax1 vector or pVax1 expressing NS5B served as negative controls. Levels of NOX1, NOX4, TGFβ1, or COX-2 mRNA were first expressed as relative to the content in the sample of mRNA of β-actin, and then normalized to the relative content of the respective mRNA in Huh7 cells transfected with the empty vector pVax1. Levels of superoxde anion were measured using DHE. All data represent the means ± S.D. from triplicate measurements done in three independent experiments. *p < 0.01; **p < 0.001.
Mentions: Our next step was to identify the molecular mechanisms by which HCV core induces the production of superoxide anion. In the cytoplasm, superoxide anion is generated by various enzymes, including the NOX family of NADPH oxidases. Crucial sources of superoxide anion in HCV-infected hepatocytes are NADPH oxidases 1 and 4 [21,22]. With this in mind, we assessed the effects of HCV core variants on the level of expression of NOX1 and NOX4 using RT-qPCR and Western blot analysis. A significant increase in both NOX1 and NOX4 mRNA and protein levels was observed in Huh7 cells expressing the full-length core(1–191) protein as well as core variants core(1–151) and core(1–36) (Figure 3, panels A–C). No up-regulation was observed in cells expressing core(37–191) or NS5B control (Figure 3A–C).

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