Construction of an expression system for HBV pseudo-viral particles Candidate No: Candidate No:

Introduction: Hepatitis B Virus Liver Specific Hepadnavirus Vaccine available > 2 billion people infected worldwide with > 350 million chronically infected patients at high risk of liver cirrhosis and hepatocellular cancer No specific treatment for patients with acute infection Need for new anti-HBV drugs Many possible liver specific viral receptors but no direct evidence

Aims Construct heterologous expression system for HBV based on HIV1 minimal three plasmid transfection (TPT) system i.e. Pseudo-type HIV1 TPT system Infect heterologous cell line expressing human liver cDNA to identify putative receptors Unambiguously link receptor viral interactions to uptake and infection

HIV1 Minimal Vector HIV1 based minimal vector HIV1 Deletion of certain regions & separation of protein coding regions Genome component Vector Gag-pol component Vector Envelope component Vector CMV  R-U5-   -Gal pGM Gag-pol CMV pGP Env CMV pEnv Three plasmid transfection (TPT) system

HIV1 Minimal Vector HIV1 based minimal vector HIV1 Deletion of certain regions & separation of protein coding regions Genome component Vector Gag-pol component Vector Envelope component Vector CMV  R-U5-   CD8 - GFP pGM “δHC” Gag-pol CMV pGP HBV Env CMV pEnv Three plasmid transfection (TPT) system Pseudo-typing

Strategy Construct heterologous expression system for HBV based on HIV1 minimal three plasmid transfection (TPT) system Pseudo-type HIV1 TPT system: Replace MA with truncated HBV core “δHC” Straight forward replacement not possible due to Lack of suitable sites for primes (PCR mutagenesis approach) Lack of convenient unique restriction enzyme sites (partial digest approach failed) HBV assembles via the ER whereas HIV buds from the plasma membrane. Need to redirect protein synthesis to the ER HIV MAHBV truncated core protein “δHC” signal target for the plasma membrane signals transport to the ER interacts with HBV envelope RNA binding region deleted (amino acids )

gag Nucleocapsid NC p7 HIV1 Minimal Vector Pseudo-typed with HBV Envelope HBV truncated core HBV envelope protein

Methods & Controls All HIV vectors supplied by Oxford Biomedica Transformations: Heat shock, XL1blue and SURE cells (E.coli) Plasmid preparation: –SS-phenol/chloroform extraction, QIAGEN and SIGMA kits RE digest at each stage to verify plasmid integrity Spectrophotometry: A260 [DNA] A260/280 - purity In-vitro transcription/translation followed by SDS- PAGE and western blotting

pGP 11kb pBSdGAG 5.5Kb XhoI EcoRV NotI δHC EcoRV NotI *Double digest EcoRVNotI Linearised vector LIGATION

δHC EcoRV NotI pBSm1GP 8.5 kb XhoI LIGATION Difficult… *small scale ligations unsuccessful transformation of SURE cells *large scale approach 1:6 vector: insert De-phosphorylated insert rather than vector

Markers Insert vector ligation mix 3kb unligated insert 8.5 kb 5.5kb unligated vector 11kb vector-vector ligation 8.5 kb ligation product Extracted and purified for transformation Difficult to extract, transformations unsuccessful Alternative approach…

PCR approach on synthetic, humanised plasmids psynGP and pHBΔC - Humanised plasmids – same protein coding regions but very different from the viral plasmid sequences 1.2kb pHBΔC psynGP primer 1 primer 2 primer 3 primer 4 500bp 700bp primer 4 primer 1 PCR 1 98°C 5 min slow cool to anneal PCR 3 Gag-Pol genesCapsid δHC Gag-Pol genes

1.2kb δHC Gag-Pol genes Capsid psynGP Mlu1 EcoRI double digest Purification Ligation

Quantitation of 500bp and 700bp PCR fragments for annealing reaction Separation of products of PCR annealing reaction MluI EcoRI digest of purified 1.2kb PCR fragment 1kb

Nested PCR approach Obtain pure insert in greater quantity Primer design Optimisation of reactions (36 trials) pHBΔC psynGP primer 1 primer 2 primer 3 Gag-Pol genesCapsid δHC primer 5 primer 4primer 6

Conclusions Closer to construction of part of HBV pseudo-typed HIV1 TPT system PCR based method adopted in favour of large scale RE digest approach. Optimisation of nested PCR achieved.

Acknowledgements Dr D Patil Dr N Ramamurthy Dr N Zitzmann All of the Virus Group With thanks to Prof. R A Dwek Thank you for your constant patience, advice and support.