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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 • 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
How does pUL38 facilitates HCMV replication and what are the interacting partners?
Construction of mutants and confirmation of normal growth Fibroblast, 0.01 PFU/cell, 0-10 dpi
Identification of pUL38 interacting proteins • 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 Fibroblast, 3 PFU/cell, 48 hrs p.i.
pUL38 interacts with the TSC2 but not with the TSC1 protein 293T, Transfection of a vector expressing pUL38, 48 hrs p.t.
TSC1 and TSC2 colocalize in infected fibroblasts BADinUL99GFP, 0.1 PFU/cell, 24 hrs p.i.
Fibroblasts expressing pUL38 are larger than normal fibroblasts
pUL38 is sufficient to prevent inhibition of the mTORC1 kinase by stress
Phosphorylation of S6 in response to stress is decreased following infection with a DUL38 virus
pUL38 interact via TSC2 with the TSC1/2 complex 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
MS & MS/MS MS/MS: Tandem Mass spectometry