construction of an expression system for hbv pseudo-viral particles candidate no: candidate no:

Construction of an expression Construction of an expression system for HBV pseudo-viral system for HBV pseudo-viral

particlesparticles

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) systemi.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 componentVector Gag-pol component

Vector

Envelope componentVector

CMV CMVR-U5- -Gal

pGM

Gag-polCMV

pGP

EnvCMV

pEnv

Three plasmid transfection (TPT) system

HIV1 Minimal Vector

HIV1 based minimal vector

HIV1

Deletion of certain regions & separation of protein coding regions

Genome componentVector Gag-pol component

Vector

Envelope componentVector

CMV CMVR-U5- CD8 - GFP

pGM

“δHC”Gag-polCMV

pGP

HBV EnvCMV

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 MA HBV 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 150-183)

gagNucleocapsid 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 integritySpectrophotometry:

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

EcoRV NotI

Linearised vector

LIGATION

δHC

EcoRV

NotI

pBSm1GP 8.5 kb

XhoI

LIGATION

Difficult…*small scale ligations unsuccessful transformation of SURE cells*large scale approach1:6 vector: insert De-phosphorylated insert rather than vector

Markers Insert vector ligation mix

3kb unligated insert

8.5 kb5.5kb 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

δHC Gag-Pol genes

1.2kb δHC Gag-Pol genes

Gag-Pol genesCapsidpsynGP

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 1kb

1kb

Nested PCR approach

Obtain pure insert in greater quantity

Primer design

Optimisation of reactions (36 trials)

pHBΔC

psynGP

primer 1primer 1

primer 2

primer 3

Gag-Pol genesCapsid

δHC

primer 5

primer 4 primer 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.