1. Gokul C Das, Ph. D Department of Medicine Baylor College of Medicine, Houston, TX 4th World Virology Congress San Antonio October 6-8, 2014

2. HCV is considered as a metabolic disease Over 170 million people world wide is infected with HCV, are at high risk for hepC, liver cirrhosis, liver cancer or type II diabetes. Induces insulin resistance (IR); IR accelerates the progression of liver disease and makes treatment difficult Needs a successful vaccine and/or an well- tolerated and cost- effective treatment strategy.

3. Recent Development in Therapy Cure for HCV by multiple oral therapy Side effects and not cost-effective $1000 /tablet (sofosbuvir, polymerase inhibitor) $84,000 (3m Course) $195,000 (per cure) Majority of HCV infection is in developing countries. Need a cost-effective and well-tolerated therapy or vaccination program

4. Questions? How does chronic HCV infection induce insulin resistance (IR)? How does IR affect the outcome of IFN-based therapeutic response? How to develop a well-tolerated and cost effective treatment strategy?

5. Hypothesis: IRS-1 phosphorylation at Ser 312 is a mediator for insulin resistance (IR) in HCV infection that involves dysregulation and interaction among metabolic pathways down stream.

6. DISRUPTION IN INSULIN SIGNALING DISTURBANCE IN GLUCOSE HOMEOSTASIS IDENTIFY mi RNA TARGETS AND PATHWAYS INVOLVED HUMANIZED MICE MODEL DEVELOPMENT OF INSULIN RESISTANCE AND MOLECULAR BASIS OF IR AND IFN RESPONSE OUR GOALS AND STRATEGY THERAPEUTIC DEVELOPMENT CHRONIC INFECTION IN LIVER CELL LINE (Huh. HCV2a)

8. Plasma Membrane PI-3K GSK-3 Glycogen Synthase(GS) Insulin Beclin 1 Bcl-2/ Bcl-XL Apoptosis IRc Insulin IRS-1/2 Glut Vesicle Autophagy Glut Akt473 Glycogen Synthesis Glucose uptake mTORC1/C2 IRc Insulin signaling pathway

9. Pathways for glucose homeostasis, insulin signaling and autophagy leading to IR in HCV infection is activated early

10. Reduced glucose uptake in infected Cells

11. MOI: 0 2 10.03.05.06.16.47 1.22 Fig. 5C

12. Huh Huh.HCV 50 100 50 100 IRS-1.06.03.99 1.01 p-IRS-1.07.04 1.22.90

14. p- Akt.36.54 1.38 1.92 p-GSK-3 α.06.03.05.04 p-GSK-3β 1.49 1.37 1.31 1.20

17. MOI: 0 1 2 Atg12-Atg5.65.78 1.30 1.38 1.32 2.61 Beclin1.15.27 1.16 1.05 1.49 2.44

18. Inhibition of autophagy reduces IRS- 1Ser312 and GS Ser641 phosphorylation

19. IRS-1 Ser312, Beclin 1 and Bcl-XL present in the same immunocomplex suggesting they interact

20. Energy Sensors AMPK and mTOR AMPK is activated during liver stress and is involved in energy regulation, autophagy and inflammation mTOR is inhibited under nutritional stress and autophagy HCV stress -----AMPK-------mTOR -----Autophagy

21. AMPK-mTOR pathway: AMPK phosphorylation and inhibition by IFNα

22. AMPK ---- 0.34 0.54 0.48 0.35 0.25 0.23 0.48 0.52 P-AMPK ---- 0.23 0.19 1.40 0.53 0.80 0.80 0.01 0.01 Ratio 0.60 0.40 2.90 1.60 3.20 3.50 0.02 0.02

23. S- 0.31.08 1.45 0.77 0.71

24. mTOR phosphorylation and inhibition by IFNα

25. Interaction of metabolic pathways with GSK-3 at the center of interaction

26. IRc PI-3K mTOR TYK2 JAK1 IRS-1/2 PI-3K Akt GSK-3 α /B Glycogen synthase IFN R1/2 Autophagy Glucose uptake IFN R1/2 TYK2 JAK1 STATs ISG Insulin IFN Protein Translation Cross talk between Insulin and IFN signaling

27. Fig.8. Strategy for inhibition of major target proteins (Akt, Gsk-3, mTOR, AMPK) and effect on the pathway

28. Summary and Discussion HCV infected cells show disruption in glucose homeostasis (reduced glucose uptake and inactivation of GS by Ser641) and it is activated within 2 weeks Insulin signaling pathway ( IRS-1-PI-3K-Akt-GSK-3) is dysregulated Autophagy is linked with IR and inhibition of autophagy inhibits IRS-1, GSK-3 beta and GS ser 641 phosphorylation Key proteins from all three major pathways interact Phosphorylation of GSK-3 beta, AMPK or mTOR are all inhibited by IFN-alpha suggesting as potential therapeutic targets. Phosphorylation of GSK-3 beta, AMPK and mTOR

29. Acknowledgement Blaine Hollinger, MD David GallardoCharles Rice, MD Wei ChenRobert Purcell, MD Cai GuangquoStan Lemon, MD Liping Bai, MDT. Wakita, MD Karina Zheng, MDGeorge Luo, MD Xianfan Huang, Ph.D Funding: Eugene B Casey Fund Carpenter fund Gulf Coast Regional Blood Center, Houston