1. From liposomes to proteomes: Investigating the cellular effects of virus and drugs Sally Latham Candidate MScRes

2. Overview BVDV model for HCV Background HCV life cycle Therapeutics Liposomal delivery of NB-DNJ Preliminary experiments Proteomics The effect of liposomes on other target cells PBMC proteomics Future work

3. Project overview HCV BVDV model Non-cytopathic What is BVDV doing to MDBKs? Cell culture NB-DNJLiposomes NB-DNJ Potential therapeutics Toxicity? Effect of liposomes on MDBK cells and PBMCs? Proteomics

4. HCV Envelope glycoprotein heterodimer Capsid Genomic + RNA Nucleocapsid Host derived membrane 50-65 nm BVDV model: Related pestivirus Non-cytopathic, cell culture in MDBKs Well studied

5. Life cycle 1. Attachment and endocytosis 2. Un-coating 3. Translation, cleavage and processing of viral proteins and replication of viral genome. 4. Budding into ER 5. Exit via Golgi/ secretory vesicles

6. Therapeutics Standard Pegylated interferon-α Immune system on: signalling - establish antiviral state Ribavirin – not antiviral alone Nucleotide analogue (~GTP)  Chain termination  error catastrophe  TH1 TH2 balance  Inosine monophosphate dehydrogenase inhibition Expensive £22k/yr Only ~50-60% effective Side effects, short term use only

7. Therapeutics Novel antiviral compounds Imino-sugar derivatives e.g. N-Butyl-deoxynojirimycin (NB-DNJ) Inhibit ER α-glucosidases I and II Prevent entry into Calnexin/Calreticulin cycle E1 and E2 can’t fold properly Retention in ER…degradation Reduction in infectious viral particle secretion Cannot achieve sufficient in vivo [NB-DNJ] (Side effects e.g. Osmotic diarrhoea)

8. Improving drug delivery pH sensitive liposomes to mediate delivery of NB-DNJ to MDBK cells infected with ncpBVDV Reduce overall imino-sugar dose Increase local [NB-DNJ] Target to the E.R.

9. Liposomes DOPE/CHEMS (DOPC/CHEMS control) Rehydration and extrusion difficulties Techniques Dessication of lipid film overnight Freeze / thaw Verified liposome size/structure by Dynamic Light Scattering – confirmed unimodal range of hydrodynamic radii.  (collaboration with A. Sonnen) Modified size exclusion chromatography protocol for separation of liposomes from external media (calcein and/or free drug)

10. Dynamic light scattering and E.M. image of DOPE/CHEMS liposomes liposomes before spin liposomes after spin 100 nm

11. Liposome characterisation DOPE/CHEMS DOPC/CHEMS

12. Liposome toxicity MTS assay data Use 30 µM [Liposome]

13. In Progress Cell culture RT-PCR Immuno-fluorescence Infectivity assay (using RT-PCR and IF) Infectivity assay merged IF image

14. Spot/band excision & Trypsin digestion Database searching Comparative Image analysis 2D-PAGE Protein sample Staining & scanning Nanospray MS/MS m/z Abundance m/z Abundance MSMS/MS Proteomics workflow

15. Proteomics Currently underway Proteomics on MDBK cells +/- BVDV, +/- liposomes CD4+ cell purification Future work: Proteomics on CD4+ PBMCs Investigating effect of liposomes

16. Naïve MDBK BVDV MDBK Possible viral proteins in BVDV infected MDBK cells Example 1 Example 2

17. Naïve MDBK BVDV MDBK Example 1Example 2 Possible PTM of cellular proteins in BVDV infection

18. Acknowledgements Thank you Nicole Bevin Steve Steph Prof R. Dwek A. Sonnen (DLS and EM) Narayan Maud Fatma Rest of the group