1. Investigation into differences between HCV permissive and non-permissive cell lines using proteomic approaches Sam Cooper Oxford University

2. Hepatitis C Virus (HCV) Around 3% of total human population infected High levels of chronicity leading ultimately to fibrosis, cirrhosis and hepatocellular carcinoma No vaccine available Current therapy (IFNalpha and ribavirin) is ineffective in a significant proportion of cases Greater understanding of the virus and its effects are clearly required

3. Cell Lines Huh7 Cell line established ~1982 Isolated from liver of 57 year old Japanese male with hepatocellular carcinoma HCV non-permissive Huh-lunet Cell line established ~2002 Derived from huh7 cells that had been cured of a viral replicon. Stably transfected with CD81 transgene. HCV permissive

4. Interferon (IFN) pathways One of the key respondants to viral attack HCV believed to disrupt some IFN signalling pathways Key pathways for antiviral effect may be downregulated in permissive cells

5. Spot/band excision & Trypsin digestion Database searching Comparative Image analysis 2D-PAGE Protein sample Staining & scanning Nanospray MS/MS SDS-PAGE Gradient gel casting m/z Abundance m/z Abundance MSMS/MS PROTEOMICS WORKFLOW Cell Culture

6. Test Gels Four test gels were run using huh7 cells A large number of distinct well defined features could be seen This meant that the lysed cells could generate appropriate 2D-PAGE gels Potentially these could be carried forward to image analysis Mw Pi

7. Cell treatment Ideally generate an endogenous interferon response pIpC chosen and introduced to cells using lipofectin Controls were untreated cells and cells treated with interferon (exogenous response) Huh7Huh-lunet UntreatedUntreated INF treated pIpC treated

8. SDS-PAGE Gel No differences were evident between the two sets of samples Loading consistency check

9. 1 st 2D gel set The pIpC treated cell lysates were run on 2D gels 27 different potential spots were seen by eye and excised for MS analysis huh7lunet Protein expression differences

10. 2 nd 2D gel set The second set of gels generated were of superior quality to the first It was determined to carry these gel images forward into analysis 1 st set2 nd set

11. Future Work Analyse second set of gels and identify spots Western blotting on gels to look for key INF pathway components Investigate the lunet cell line permissiveness for other viruses such as EMCV and HSV2 Additional experiment

12. Investigating treatment effects on huh-lunet Untreated control NB-DNJ NB-DGJ NN-DNJ NN-DGJ Liposome control Liposomes Liposomes + NB-DNJ

13. Thanks to: Prof. Dwek Dr. Zitzmann Bevin Gangadharan Maud Michelet and all the Antiviral Drug Discovery Unit