Transcript Construction of an expression system for HBV pseudo-viral particles  Candidate No: Introduction: Hepatitis B Virus • Liver Specific Hepadnavirus • Vaccine available • > 2 billion.

Construction of an expression
system for HBV pseudo-viral
particles

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
Three plasmid
transfection
(TPT) system
HIV1
Deletion of certain regions
& separation of protein coding regions
HIV1 based minimal vector
Genome component
Vector
CMV
dR-U5-y CMV
pGM
b-Gal
Gag-pol component
Vector
CMV
Gag-pol
Envelope component
Vector
CMV
Env
pGP
pEnv
HIV1 Minimal Vector
Three plasmid
transfection
(TPT) system
HIV1
Deletion of certain regions
& separation of protein coding regions
Pseudo-typing
HIV1 based minimal vector
Genome component
Vector
CMV
dR-U5-y CMV
pGM
CD8 - GFP
Gag-pol component
Vector
CMV
“δHC”Gag-pol
Envelope component
Vector
CMV
HBV Env
pGP
pEnv
Strategy
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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)
HIV1 Minimal Vector Pseudo-typed with HBV Envelope
HBV envelope
protein
HBV
truncated core
gag
Nucleocapsid NC
p7
Methods & Controls
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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 SDSPAGE and western blotting
EcoRV
EcoRV
NotI
NotI
XhoI
pGP
11kb
δHC
pBSdGAG
5.5Kb
*Double digest
EcoRV
NotI
Linearised vector
LIGATION
EcoRV
XhoI
δHC
NotI
pBSm1GP
8.5 kb
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
11kb vector-vector ligation
8.5 kb
5.5kb unligated vector
3kb unligated insert
8.5 kb ligation product
Extracted and purified
for transformation
Difficult to extract, transformations unsuccessful
Alternative approach…
PCR approach on synthetic, humanised plasmids
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psynGP and pHBΔC - Humanised plasmids – same protein coding regions but
very different from the viral plasmid sequences
primer 1
pHBΔC
δHC
primer 2
PCR 1
primer 3
psynGP
Capsid
Gag-Pol genes
primer 4
500bp
700bp
98°C 5 min slow
cool to anneal
primer 1
primer 4
PCR 3
1.2kb
δHC
Gag-Pol genes
1.2kb
δHC
psynGP
Gag-Pol genes
Capsid
Gag-Pol genes
Mlu1 EcoRI double digest
Purification
Ligation
1kb
1kb
Quantitation of 500bp and
700bp PCR fragments for
annealing reaction
MluI EcoRI digest of
purified 1.2kb PCR
fragment
Separation of products of
PCR annealing reaction
1kb
Nested PCR approach
Obtain pure insert in greater quantity
Primer design
Optimisation of reactions (36 trials)
primer 5
primer 1
pHBΔC
δHC
primer 2
primer 3
psynGP
Capsid
Gag-Pol genes
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
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Dr D Patil
Dr N Ramamurthy
Dr N Zitzmann
All of the Virus Group
Thank you for your
constant patience,
advice and support.
• With thanks to Prof. R A Dwek