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HCVM
• Member of -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
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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
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How does pUL38 facilitates HCMV replication and what are the
interacting partners?
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Construction of mutants and confirmation of normal growth
Fibroblast, 0.01 PFU/cell, 0-10 dpi
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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
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pUL38 interacts with the TSC1/2 protein complex
Fibroblast, 3 PFU/cell, 48 hrs p.i.
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pUL38 interacts with the TSC2 but not with the TSC1 protein
293T, Transfection of a vector expressing pUL38, 48 hrs p.t.
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TSC1 and TSC2 colocalize in infected fibroblasts
BADinUL99GFP, 0.1 PFU/cell, 24 hrs p.i.
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Fibroblasts expressing pUL38 are larger than normal fibroblasts
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pUL38 is sufficient to prevent inhibition of the mTORC1 kinase by stress
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Phosphorylation of S6 in response to stress is decreased following infection
with a UL38 virus
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Summary
• 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
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pUL38 affects cellular pathways that communicate via TSC1/2
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Thank you for your attention!
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Activation of the TSC1/2 complex
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MS & MS/MS
MS/MS: Tandem Mass spectometry