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HCVM. Member of b -herpesvirus family >200 ORFs Genes are expressed in a regulated cascade Some fulfill regulatory functions pUL38 expression during early phase pUL38 blocks apoptosis. http://www.abbottdiagnostics.co.uk. Tuberous Sclerosis Complex. Autosomal dominant disorder

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HCVM

  • Member of b-herpesvirus family

  • >200 ORFs

  • Genes are expressed in a regulated cascade

  • Some fulfill regulatory functions

  • pUL38 expression during early phase

  • pUL38 blocks apoptosis

http://www.abbottdiagnostics.co.uk


Tuberous sclerosis complex
Tuberous Sclerosis Complex

  • Autosomal dominant disorder

  • Characterized by benign tumors

     Due to inactivating mutations in TSC1 or TSC2

  • Conserved signaling pathway that regulates cell growth

  • TSC1/TSC2 functions as GAP towards Rheb



Construction of mutants and confirmation of normal growth
Construction of mutants and confirmation of normal growth interacting partners?

Fibroblast, 0.01 PFU/cell, 0-10 dpi


Identification of pul38 interacting proteins
Identification of pUL38 interacting proteins interacting partners?

  • pUL38 is expressed under proper kinetics and at normal levels

  • Isolation by immuno- affinity purification

  • Separation by gel electrophoresis

  • Identification by sequential MS and MS/MS analysis

     ~ 50 target proteins


Pul38 interacts with the tsc1 2 protein complex
pUL38 interacts with the TSC1/2 protein complex interacting partners?

Fibroblast, 3 PFU/cell, 48 hrs p.i.


Pul38 interacts with the tsc2 but not with the tsc1 protein
pUL38 interacts with the TSC2 but not with the TSC1 protein interacting partners?

293T, Transfection of a vector expressing pUL38, 48 hrs p.t.


Tsc1 and tsc2 colocalize in infected fibroblasts
TSC1 and TSC2 colocalize in infected fibroblasts interacting partners?

BADinUL99GFP, 0.1 PFU/cell, 24 hrs p.i.




Phosphorylation of s6 in response to stress is decreased following infection with a d ul38 virus
Phosphorylation of S6 in response to stress is decreased following infection with a DUL38 virus


Summary

pUL38 interact via TSC2 with the TSC1/2 complex following infection with a

Ectopic expression of pUL38 in fibroblasts increased their size

Interaction of pUL38 with TSC1/2 blocked its ability to regulate mTORC1 in response to stress outside the context of infection

In the presence of pUL38, AMPK stimulation did not block phosphorylation of rpS6

Summary



Thank you for your attention

Thank you for your attention! following infection with a


Activation of the tsc1 2 complex
Activation of the TSC1/2 complex following infection with a


Ms ms ms
MS & MS/MS following infection with a

MS/MS: Tandem Mass spectometry