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