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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

Structure and expression of HCV core variants used to induce oxidative stress and oxidative stress response. (A) Schematic representation of HCV core and its truncated forms: the N-terminal fragment aa 1-36, the N-terminally deleted fragment encompassing aa 37–191 and the C-terminally deleted variant encompassing core aa 1–151, dubbed core(1–191), core(1–36), core(37–191) and core(1–151), respectively. Charts below represent the known elements of core structure: structural domains [24], domains responsible for RNA-binding [24], oligomerization [24], localization to lipid droplets [25]; and binding to host proteins STAT1 [36], DDX3 [37], LT-betaR [35]; as well as the position of the helix-loop-helix motif, nuclear localization (NLS) and nuclear export signals (NES) [34]; (B) Intracellular localization in Huh7 cells of the full-length and truncated HCV core variants (depicted on the left): staining with primary rabbit anti-core and secondary anti-rabbit antibodies conjugated to FITC or TRITC (panel I), nuclear staining with Hoechst 33258 (panel II), overlay of panels I and II (panel III) (see Materials and Methods Section for details).
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viruses-07-02745-f001: Structure and expression of HCV core variants used to induce oxidative stress and oxidative stress response. (A) Schematic representation of HCV core and its truncated forms: the N-terminal fragment aa 1-36, the N-terminally deleted fragment encompassing aa 37–191 and the C-terminally deleted variant encompassing core aa 1–151, dubbed core(1–191), core(1–36), core(37–191) and core(1–151), respectively. Charts below represent the known elements of core structure: structural domains [24], domains responsible for RNA-binding [24], oligomerization [24], localization to lipid droplets [25]; and binding to host proteins STAT1 [36], DDX3 [37], LT-betaR [35]; as well as the position of the helix-loop-helix motif, nuclear localization (NLS) and nuclear export signals (NES) [34]; (B) Intracellular localization in Huh7 cells of the full-length and truncated HCV core variants (depicted on the left): staining with primary rabbit anti-core and secondary anti-rabbit antibodies conjugated to FITC or TRITC (panel I), nuclear staining with Hoechst 33258 (panel II), overlay of panels I and II (panel III) (see Materials and Methods Section for details).

Mentions: We and others have previously shown that expression of HCV core protein in eukaryotic cells induces oxidative stress and activates the Nrf2/ARE antioxidant defense pathway by several, possibly independent, mechanisms [15,16,22]. Mechanistic studies were, however, hampered by simultaneous involvement of HCV core in several ROS-producing and ROS-scavenging processes. To delineate them, we designed a panel of truncated forms of HCV core protein that would activate only a minimum, ideally one, of the pathways. The panel included a peptide representing the N-terminus of the core protein (aa 1–36) with two nuclear localization signals [34] previously implicated in various protein-protein interactions (Figure 1A) [35,36,37]. The other two truncated forms of HCV core were devoid of the N-terminal 36 amino acids as core(37–191), or of the C-terminal 40 amino acids as core(1–151) (note the deletion of ER retention sequence). HCV NS5B served as a negative control, since our previous data demonstrated that this protein has no effect on the production of ROS even expressed to high levels [15].


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)

Structure and expression of HCV core variants used to induce oxidative stress and oxidative stress response. (A) Schematic representation of HCV core and its truncated forms: the N-terminal fragment aa 1-36, the N-terminally deleted fragment encompassing aa 37–191 and the C-terminally deleted variant encompassing core aa 1–151, dubbed core(1–191), core(1–36), core(37–191) and core(1–151), respectively. Charts below represent the known elements of core structure: structural domains [24], domains responsible for RNA-binding [24], oligomerization [24], localization to lipid droplets [25]; and binding to host proteins STAT1 [36], DDX3 [37], LT-betaR [35]; as well as the position of the helix-loop-helix motif, nuclear localization (NLS) and nuclear export signals (NES) [34]; (B) Intracellular localization in Huh7 cells of the full-length and truncated HCV core variants (depicted on the left): staining with primary rabbit anti-core and secondary anti-rabbit antibodies conjugated to FITC or TRITC (panel I), nuclear staining with Hoechst 33258 (panel II), overlay of panels I and II (panel III) (see Materials and Methods Section for details).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488712&req=5

viruses-07-02745-f001: Structure and expression of HCV core variants used to induce oxidative stress and oxidative stress response. (A) Schematic representation of HCV core and its truncated forms: the N-terminal fragment aa 1-36, the N-terminally deleted fragment encompassing aa 37–191 and the C-terminally deleted variant encompassing core aa 1–151, dubbed core(1–191), core(1–36), core(37–191) and core(1–151), respectively. Charts below represent the known elements of core structure: structural domains [24], domains responsible for RNA-binding [24], oligomerization [24], localization to lipid droplets [25]; and binding to host proteins STAT1 [36], DDX3 [37], LT-betaR [35]; as well as the position of the helix-loop-helix motif, nuclear localization (NLS) and nuclear export signals (NES) [34]; (B) Intracellular localization in Huh7 cells of the full-length and truncated HCV core variants (depicted on the left): staining with primary rabbit anti-core and secondary anti-rabbit antibodies conjugated to FITC or TRITC (panel I), nuclear staining with Hoechst 33258 (panel II), overlay of panels I and II (panel III) (see Materials and Methods Section for details).
Mentions: We and others have previously shown that expression of HCV core protein in eukaryotic cells induces oxidative stress and activates the Nrf2/ARE antioxidant defense pathway by several, possibly independent, mechanisms [15,16,22]. Mechanistic studies were, however, hampered by simultaneous involvement of HCV core in several ROS-producing and ROS-scavenging processes. To delineate them, we designed a panel of truncated forms of HCV core protein that would activate only a minimum, ideally one, of the pathways. The panel included a peptide representing the N-terminus of the core protein (aa 1–36) with two nuclear localization signals [34] previously implicated in various protein-protein interactions (Figure 1A) [35,36,37]. The other two truncated forms of HCV core were devoid of the N-terminal 36 amino acids as core(37–191), or of the C-terminal 40 amino acids as core(1–151) (note the deletion of ER retention sequence). HCV NS5B served as a negative control, since our previous data demonstrated that this protein has no effect on the production of ROS even expressed to high levels [15].

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