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

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

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

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

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

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

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)

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.

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)

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

Liposome characterisation DOPE/CHEMS DOPC/CHEMS

Liposome toxicity MTS assay data Use 30 µM [Liposome]

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

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

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

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

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

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