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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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Activation of the TSC1/2 complex
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MS & MS/MS MS/MS: Tandem Mass spectometry
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