investigation into differences between hcv permissive and non-permissive cell lines using proteomic...
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Investigation into differences Investigation into differences between HCV permissive and between HCV permissive and
non-permissive cell lines non-permissive cell lines using proteomic approachesusing proteomic approaches
Sam CooperSam Cooper
Oxford Oxford UniversityUniversity
Hepatitis C Virus (HCV)Hepatitis C Virus (HCV)
•Around 3% of total human population infected•High levels of chronicity leading ultimately to fibrosis, cirrhosis and hepatocellular carcinoma•No vaccine available•Current therapy (IFNalpha and ribavirin) is ineffective in a significant proportion of cases•Greater understanding of the virus and its effects are clearly required
Cell LinesCell Lines
Huh7Huh7
Cell line established Cell line established ~1982~1982
Isolated from liver of Isolated from liver of 57 year old 57 year old Japanese male with Japanese male with hepatocellular hepatocellular carcinomacarcinoma
HCV non-permissiveHCV non-permissive
Huh-lunetHuh-lunet
Cell line established Cell line established ~2002~2002
Derived from huh7 Derived from huh7 cells that had been cells that had been cured of a viral cured of a viral replicon. Stably replicon. Stably transfected with transfected with CD81 transgene.CD81 transgene.
HCV permissiveHCV permissive
Interferon (IFN) pathwaysInterferon (IFN) pathways
One of the key One of the key respondants to viral respondants to viral attackattack
HCV believed to HCV believed to disrupt some IFN disrupt some IFN signalling pathwayssignalling pathways
Key pathways for Key pathways for antiviral effect may antiviral effect may be downregulated in be downregulated in permissive cellspermissive cells
Spot/band excision & Trypsin digestion
Database searching
Comparative Image analysis
2D-PAGE
Protein sample
Staining & scanning
Nanospray MS/MS
SDS-PAGE
Gradient gel casting
m/z
Ab
un
dan
ce
m/z
Ab
un
dan
ce
MS MS/MS
PROTEOMICS WORKFLOW
Cell Culture
Test GelsTest Gels
Four test gels were run Four test gels were run using huh7 cellsusing huh7 cellsA large number of A large number of distinct well defined distinct well defined features could be seenfeatures could be seenThis meant that the This meant that the lysed cells could lysed cells could generate appropriate generate appropriate 2D-PAGE gels2D-PAGE gelsPotentially these could Potentially these could be carried forward to be carried forward to image analysisimage analysis
Mw
Pi
Cell treatmentCell treatment
Ideally generate an Ideally generate an endogenous interferon endogenous interferon responseresponse
pIpC chosen and pIpC chosen and introduced to cells using introduced to cells using lipofectinlipofectin
Controls were untreated Controls were untreated cells and cells treated cells and cells treated with interferon with interferon (exogenous response)(exogenous response)
Huh7Huh7 Huh-lunetHuh-lunet
UntreatedUntreated UntreatedUntreated
INF INF treatedtreated
INF INF treatedtreated
pIpC pIpC treatedtreated
pIpC pIpC treatedtreated
SDS-PAGE GelSDS-PAGE Gel
No differences were No differences were evident between the evident between the two sets of samples two sets of samples
Loading consistency Loading consistency checkcheck
<------huh7------><-----Lunet----->
11stst 2D gel set 2D gel set
The pIpC treated cell lysates were run on 2D gelsThe pIpC treated cell lysates were run on 2D gels
27 different potential spots were seen by eye and 27 different potential spots were seen by eye and excised for MS analysisexcised for MS analysis
huh7 lunet
Protein expression differences
22ndnd 2D gel set 2D gel set
The second set of gels generated were of superior The second set of gels generated were of superior quality to the firstquality to the firstIt was determined to carry these gel images forward It was determined to carry these gel images forward into analysisinto analysis
1st set 2nd set
Future WorkFuture Work
Analyse second set of gels and identify Analyse second set of gels and identify spotsspots
Western blotting on gels to look for key Western blotting on gels to look for key INF pathway componentsINF pathway components
Investigate the lunet cell line Investigate the lunet cell line permissiveness for other viruses such as permissiveness for other viruses such as EMCV and HSV2EMCV and HSV2
Additional experimentAdditional experiment
Additional experimentAdditional experiment
Investigating treatment effects on huh-lunetInvestigating treatment effects on huh-lunet
Untreated controlUntreated control
NNB-DNJB-DNJ
NNB-DGJB-DGJ
NNN-DNJN-DNJ
NNN-DGJN-DGJ
Liposome controlLiposome control
LiposomesLiposomes
Liposomes + Liposomes + NNB-DNJB-DNJ
Thanks to:Thanks to:
Prof. DwekProf. Dwek
Dr. ZitzmannDr. Zitzmann
Bevin GangadharanBevin Gangadharan
Maud MicheletMaud Michelet
and all the Antiviral Drug Discovery Unitand all the Antiviral Drug Discovery Unit