HCV activates oxidative stress defense system and suppresses polyamine biosynthesis

1. Engelhardt Institute of Molecular Biology Russian Academy of Sciences HCV activates oxidative stress defense system and suppresses polyamine biosynthesis Smirnova Olga

2. HCV induces oxidative stress: mechanisms and consequences • HCV-induced oxidative stress can contribute to: • Liver fibrosis • “Non-Vogelstein type” cancerogenesis • Interferon  resistance in chronic hepatitis C patients • Insulin resistance • Alteration of iron homeostasis via inhibition of hepcidin expression • HCV causes oxidative stress by multiple mechanisms: • Core and NS5A proteins alter calcium homeostasis resulting in accumulation of Reactive Oxygen Species (ROS) • E1, E2, and NS4B proteins induce ER-stress which can also lead to activation of ER oxidoreductases and to accumulation of reactive oxygen species (ROS) • HCV proteins activate NADPH oxidase 4 (Nox4) expression

3. р Nrf2 Regulation of antioxidant defense system Phase II detoxyfying enzymes Nonhazardous products ROS [ROS]* antioxidants PRE (Polyamine Response Element) ARE (Antioxidant Response Element) р Nrf2 Keap1 Nrf2 kinases inhibitors ER stress NS4В PERK NS5A pcDNA3.1(+) Capsid protein pGL3-promoter ARE Phase II enzymes luciferase luciferase РRE SSAT Spermine/spermidine N1-acetyltransferase pP-ARE pP-PRE

4. HCV proteins induce oxidative stress

5. HCV proteins activate ARE-dependent protein expression Impact of HCV proteins on ARE-dependent luciferase expression Up-regulation of HO-1 and Nqo1 gene expression by HCV proteins

6. HCV proteins activate ARE-dependent transcription by ROS-dependent and –independent mechanisms PDTC (pyrrolidine dithiocarbamate) acts as ROS scavenger

7. HCV proteins induce ARE-luciferase expression via PKC in a ROS-dependent, and by PI3K and CK2 in a ROS-independent manner. Wo (Wortmannin) – PI3K inhibitor Ro (Ro31-8220) – PKC inhibitor DRB – CK2 inhibitor

8. Effect of protein kinase inhibitors and PDTC on HO-1/Nqo1 gene expression, and Nrf2 localization

9. Conclusions • HCV proteins induced strong up-regulation of antioxidant defense system that might mitigate harmful effects of HCV-induced oxidative stress during acute stage of hepatitis C • Core, E1, E2, NS4B, and NS5A activate Nrf2/ARE pathway through three independent mechanisms • All five proteins induced accumulation of reactive oxygen species (ROS) which activated protein kinaseC • Core and NS5A proteins induced ROS-independent activation of casein kinase 2 and phosphoinositide-3 kinase • The effects of NS4B, E1, and E2 were presumably mediated via ROS-independent PERK pathway

10. Acknowledgements Sergey Kochetkov lab (EIMB)Karolinska Institutet (Sweden) Alex Ivanov Maria Isaguliants Olga Ivanova Alexey Khomutov A.I. Virtanen Institute (Finland) Tuomo Keinänen Mervi Hyvonen The project was supported by: • Russian Foundation for Basic Research • Grant of President of Russian Federation

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